Publications

Book Chapters

Book chapters authored or coauthored by members of the Laboratory for Fluorescence Dynamics (LFD) from 1987 to present. Access to most PDF documents is password protected due to copyright restrictions. Reprints can be requested from the authors.

2016

di Rienzo C, Gratton E, Beltram F, Cardarelli F.

Super-Resolution in a standard microscope: from fast fluorescence imaging to molecular diffusion laws in live cells.

Living systems establish local steady-state conditions by maintaining a complex landscape of precisely regulated interactions, which govern the spatial distribution of their molecular constituents. In this regard, fluorescence fluctuation microscopy encompasses a diversified arsenal of analysis tools that provide a quantitative link between cell structural organization and the underlying molecular dynamics. A spatial or temporal analysis of the fluctuating fluorescence signal arising from suitably labeled reporters allows noninvasive measurements of molecular and biochemical parameters such as concentration, diffusion coefficients, binding constants, degree of oligomerization, and so on directly within nonperturbed, live samples. Thus, within the constraints imposed by the requirement of fluorescence labeling, fluctuation microscopy has the potential to add a dynamic molecular dimension to standard fluorescence imaging in vivo. In particular, we shall discuss a fluorescence fluctuation-based method that makes it possible to probe the actual molecular “diffusion law” directly from imaging, in the form of a mean square ... [truncated at 150 words]

Gratton E.

Measurements of fluorescence decay time by the frequency domain method.

Perspectives on Fluorescence: A Tribute to Gregorio Weber (Springer Series on Fluorescence). By DM Jameson (Editors). Springer Berlin Heidelberg, 2016.

Among the many contributions of Gregorio Weber to science and technology, the development of frequency domain technology in his lab in 1969 has caused a deep controversy, dividing scientist that will refuse using anything but the frequency domain approach to measure the fluorescence decay from the other camp that simply refuse anything but the time-correlated single photon counting (TCSPC) approach. Although at the time of the major contribution of Gregorio Weber and Richard Spencer in 1969, the TCSPC method was not yet invented, the basic controversy “frequency domain vs time domain” in the field continues today. We have made progress both in the scientific understanding and in describing the technical differences between the two approaches; still it is interesting how scientists continue to be divided. As for many of the contributions of Gregorio Weber that stirred controversy, I would like to mention a common theme of my conversations with Dr. ... [truncated at 150 words]

Cardarelli F, Gratton E.

Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time.

Perspectives on Fluorescence: A Tribute to Gregorio Weber (Springer Series on Fluorescence). By DM Jameson (Editors). Springer Berlin Heidelberg, 2016.

The fundamental unit of biology is unarguably the cell. Thus, as we move forward in our understanding of the processes occurring in the cell, it is crucial to reflect on how much of the cell biophysics remains unexplained or unknown. A ubiquitous observation in cell biology is that the translational motion of molecules within the intracellular environment is strongly suppressed as compared to that in dilute solutions. By contrast, molecular rotation is not affected by the same environment, indicating that the close proximity of the molecule must be aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding, but with expectations that depend on the nanoscale organization assigned to crowding agents. A satisfactory experimental discrimination between possible scenarios has remained elusive due to the lack of techniques to explore molecular diffusion at the appropriate spatiotemporal scale in the 3D-intracellular environment. Here we discuss ... [truncated at 150 words]

INTRODUCTION There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluorescent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their lipid composition, it is more useful to use a single probe that can report on the specific properties of the membrane icrodomains, independently of the probe segregation in one specific domain and location in the cell. One fluorescent probe that has been successfully used for this purpose is the lipophilic probe Laurdan (2-dimethylamino-6-lauroylnaphthalene), originally synthesized by Weber and Farris.1 Different membrane environments produce marked changes both in the spectrum and in the fluorescence lifetime of Laurdan. The sensitivity of the emission spectrum ... [truncated at 150 words]

Digman MA.

Fluctuation spectroscopy methods for the analysis of membrane processes.

INTRODUCTION In the field of membrane lipid and protein dynamics, fluorescence recovery after photobleaching (FRAP) and single-particle tracking methods have provided ample evidence that lipids and proteins could have their motion restricted by interaction with other lipids and with the cell cytoskeleton.1–8 Fluorescence correlation spectroscopy (FCS) is a relatively new method in this field that has received particular attention recently because of the possible combination with super-resolution microscopy methods.9–14 In this context, the use of very small volumes of excitation or variable volumes of excitation15 was considered necessary to unravel to transport of molecules at the nanoscale. However, most of the FCS studies done so far are based on the original idea of measuring temporal correlation at a single point in the membrane. Measuring a single location in the membrane is restrictive since the temporal fluctuations at one point cannot reveal local microstructures or the anisotropic molecular transport in membranes. ... [truncated at 150 words]

Golfetto O, Hinde E, Gratton E.

The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells.

In this method paper we describe the spectral phasor analysis applied to Laurdan emission for the assessment of the fluidity of different membranes in live cells. We first introduce the general context and then we show how to obtain the spectral phasor from data acquired using a commercial microscope.

The phasor approach to fluorescence lifetime imaging (FLIM) is emerging as a practical method for data visualization and analysis. The main feature of the phasor approach is that it is a “fit-free” analysis tool that provides quantitative results about mixtures of fluorophores, Förster resonant energy transfer (FRET), and autofluorescence.

2013

Gratton E, Stakic M, Digman MA.

Raster image correlation spectroscopy and number and brightness analysis.

The raster image correlation spectroscopy (RICS) and number and molecular brightness (N&B) methods are used to measure molecular diffusion in complex biological environments such as the cell interior, detect the formation of molecular aggregates, establish the stoichiometry of the aggregates, spatially map the number of mobile molecules, and quantify the relative fraction of molecules participating in molecular complexes. These methods are based on correlation of fluorescence intensity fluctuations from microscope images that can be measured in a conventional laser-scanning confocal microscope. In this chapter, we discuss the mathematical framework used for data analysis as well as the parameters need for data acquisition. We demonstrate the information obtainable by the N&B method using simulation in which different regions of an image have different numbers of interacting molecules. Then, using an example of two interacting proteins in the cell, we show in a real case how the RICS and N&B analyses work ... [truncated at 150 words]

Single-particle and single molecules techniques have become essential tools in the fields of Biophysics and Cell Biology. One of the main reasons of the strong impact of these techniques is that they provide crucial information that is average out in traditional ensemble methods. Amongst these new techniques, single particle tracking (SPT) constituted a remarkable new tool to study dynamics of biological processes. In this chapter, we briefly describe most common techniques used for tracking particles and focus in recent advances in the field of microscopy that resulted in an improvement of these methods. We discuss different strategies followed to obtain information regarding the axial position of the particle in image-based tracking approaches and describe in detail a routine we have designed to achieve three dimensional (3D) tracking with a laser scanning microscope. Finally, we show the application of this technique to the study of chromatin dynamics in interphase cells to ... [truncated at 150 words]

There is a series of outstanding questions regarding the detection of brain activity in measurements made at the head surface and regarding the origin of the observed changes in the optical parameters of tissues. In this chapter we review the information generated so far and discuss the evidence available about the origin of the effects observed. Because the field is still controversial and rapidly advancing, we mainly focus on our own work and a few other studies that we chose to illustrate our opinions...

Laurdan is a fluorescent molecule that detects changes in membrane phase properties through its sensitivity to the polarity of the environment in the bilayer. Polarity changes are shown by shifts in the Laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). This technique was originally developed to be used in a cuvette fluorometer. With the development of twophoton microscopy, Laurdan GP has evolved into a technique capable of spatially resolve micro-domains of different solvent penetration. We discuss here the basic concepts, instrumentation and experimental considerations when transferring the GP technique from the cuvette to the microscope. We also discuss examples of Laurdan GP in membrane model systems using both cuvette and microscope approaches to compare the two methodologies.

The authors developed a technique to apply high hydrostatic pressure to giant unilamellar vesicles and to directly observe the consequent structural changes with two-photon fluorescence microscopy imaging using high numerical aperture oil-immersion objectives. The data demonstrate that high pressure has a dramatic effect on the shape of the vesicles, and both fluidity and homogeneity of the membrane.

Digman MA, Gratton E.

The RICS method: measurement of fast dynamics in cells with the laser scanning microscope.

Single point fluctuation correlation spectroscopy (FCS) is an established technique to study diffusion and chemical equilibria in solution. It has limitations when applied to the cell interior. A major difficulty is that the movements of the entire cell or of cellular components are difficult to separate and filter out from the fast dynamics of the molecules moving in the cell. It is the study of these fast dynamics that helps us in understanding molecular interactions. Scanning FCS, in which the laser beam is moved in a circular orbit, provides the fluctuation amplitude and dynamics at many points simultaneously. It can be used to infer cell movement, but has limitations in the time scales accessible. Image correlation spectroscopy, an alternative technique in which the entire field of view is analyzed at once, has the potential to provide detailed maps of the dynamics in a cell, but it suffers from limitations imposed ... [truncated at 150 words]

Laser assisted confocal microscopy has made a lot of progress over the past few years. Laser systems have become more modular and compact. There is an ever-increasing number of available laser excitation lines as well as an improvement in user friendliness and ease of use. At the same time, expansion of web resources has provided easy access to a wealth of information. Our goal is both to aid the experienced and novice microscopist in quickly locating and sorting through the relevant laser information and to provide a means of avoiding common problems and pitfalls in the use of laser excitation in the various fluorescence techniques such as fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), fluorescence loss in photobleaching (FLIP), fluorescence recovery after photobleaching (FRAP), optical coherence tomography (OCT), second harmonic generation (SHG), single molecule detection (SMD), and single particle tracking (SPT). In this chapter we describe the characteristic ... [truncated at 150 words]

Fluorescence Correlation Spectroscopy (FCS) was first introduced by Elson, Madge and Webb (1-5) for studying the binding process between ethydium bromide and DNA. When the ethydium dye binds to DNA its fluorescence quantum yield changes by a large factor. It is essentially not fluorescent when free in solution and it becomes strongly fluorescent when bound to double strand DNA. Although the processes are very different in nature, the instrumentation used for the FCS experiment is derived from dynamic light scattering. There are however major differences between dynamic light scattering and FCS...

SummaryThis chapter discusses practical features and guidelines for Fluorescence lifetime imaging (FLI) and Förster resonance energy transfer (FRET) that maybe helpful in reading the literature and in interpreting FLI and FRET measurements of complex biological systems. Several reviews are already available (Clegg and Schneider, 1996; [Clegg, et al, 1996] and [Clegg, et al, 2003]; Periasamy et al., 1996; [So, et al, 1996] and [So, et al, 1998]; Schneider and Clegg, 1997; Gadella et al., 2001; Cubeddu et al., 2002; Hink et al., 2002; Peter and Ameer-Beg, 2004). However, we feel that the general reader and aspiring user of FLI with applications to FRET would benefit from a concise, coherent presentation of fundamental aspects of these measurements and interpretations of the data. We will not include particular results from a biological system, nor specifics of new instrumentation. Instead, we focus on basic descriptions of the photophysical measurements and the general characteristics ... [truncated at 150 words]

The applications of Fluorescence Resonance Energy Transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. Our understanding of photosynthesis is tightly coupled to our understanding of the transfer of captured energy from the absorption of photons, and following the energy flow through the complex maize of chemical reactions utilizing this energy. Many of these steps involve resonance energy transfer. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation. This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis. Many publications appear weekly using FRET and most of the applications use FRET as a spectroscopic research tool. Photosynthesis holds ... [truncated at 150 words]

Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations ... [truncated at 150 words]

Gratton E, Fantini S.

Reflectance and transmittance spectroscopy.

Laser and Current Optical Techniques in Biology (Comprehensive Series in Photochemical & Photobiological Sciences). By G Palumbo and R Pratesi (Editors). Royal Society of Chemistry, pp. 211-258, 2005. ISBN: 9780854043217

This chapter describes several approaches to the optical study of biological tissue using reflectance and transmittance spectroscopy. This topic has spurred significant research efforts as a result of the relevant physiological and metabolic information provided by the optical data, in conjunction with the safe, non-invasive, and costeffective optical approach to the study of tissue. We give a brief historical introduction in Section 11.1, followed by a description of the optical absorption and scattering properties of tissue in Section 11.2. Section 11.3 is devoted to 1 continuous-wave (CW) techniques, which can be applied to the study of relatively superficial tissue layers (as is the case for diffuse reflectance imaging and localized measurements using short separations between the illuminating and collecting optodes), as well as deep tissues on the basis of a modified Beer-Lambert law, transport theory, or diffusion theory. The latter model is commonly employed in time-domain and frequency-domain techniques, which ... [truncated at 150 words]

Hanson KM, Clegg RM.

Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis.

Two-photon fluorescence microscopy is used to detect ultraviolet-induced reactive oxygen species (ROS) in the epidermis and the dermis of ex vivo human skin and skin equivalents. Skin is incubated with the nonfluorescent ROS probe dihydrorhodamine, which reacts with ROS such as singlet oxygen and hydrogen peroxide to form fluorescent rhodamine-123. Unlike confocal microscopic methods, two-photon excitation provides depth penetration through the epidermis and dermis with little photodamage to the sample. This method also provides submicron spatial resolution such that subcellular areas that generate ROS can be detected. In addition, comparative studies can be made to determine the effect of applied agents (drugs, therapeutics) upon ROS levels at any layer or cellular region within the skin.

2004

Palumbo G, Pratesi R, Gratton E, Fantini S, Häder DP, Jori G.

Reflectance and transmittance spectroscopy.

Lasers and Current Optical Techniques in Biology (Comprehensive Series in Photochemical & Photobiological Sciences). By G Palumbo and R Pratesi (Editors). Royal Society of Chemistry, pp. 213-247, 2004. ISBN: 9780854043217

This chapter describes several approaches to the optical study of biological tissue using reflectance and transmittance spectroscopy. This topic has spurred significant research efforts as a result of the relevant physiological and metabolic information provided by the optical data, in conjunction with the safe, non-invasive, and costeffective optical approach to the study of tissue. We give a brief historical introduction in Section 1 1.1, followed by a description of the optical absorption and scattering properties of tissue in Section 11.2. Section 11.3 is devoted to continuous-wave (CW) techniques, which can be applied to the study of relatively superficial tissue layers (as is the case for diffuse reflectance imaging and localized measurements using short separations between the illuminating and collecting optodes), as well as deep tissues on the basis of a modified Beer-Lambert law, transport theory, or diffusion theory. The latter model is commonly employed in time-domain and frequency-domain techniques, which ... [truncated at 150 words]

Three different methods of fluorescence-lifetime imaging for microscopy are presented along with some examples of their use. All three methods use the frequency-domain heterodyning technique to collect lifetime data. Because of the nature of the data collection technique, these instruments can measure the correct lifetime even when the sample undergoes strong photobleaching. Each instrument has unique capabilities that complement the others.The first microscopic-lifetime imaging technique uses a fast charge-coupled device (CCD) camera and a gated image intensifier. The camera system collects an entire lifetime image in parallel in only a few seconds. This microscope is well suited to kinetic studies of intracellular lifetime changes. The other two techniques use scanned laser source to collect sequential lifetime information pixel by pixel. One microscope uses two-photon excitation to achieve three-dimensional, confocal-like imaging without using detection pinholes. Two-photon excitation also limits the effects of out-of-plane photodamage of the sample. The second laser-scanning microscope ... [truncated at 150 words]

The aim of this chapter is to describe, in detail, the design, application, and "philosophy" behind the current generation of global analysis programs. The sections of this chapter can be summarized as follows:Section 1: Introduction to time-resolved fluorescence data, some experimental techniques, and some typical examples of how previous works have benefitted from global analysis.Section 2: Historical overview of how the emphasis of global analysis has evolved from one of multi-dimensional curve fitting to that of multi-dimensional physical model evaluation.Section 3: General elements required to perform a global analysis of distributed and discrete models. The basic equations of the compartmental approach are examined and the systems theory view of photophysical events is described.Section 4: In-depth example of the "inner-workings" of the general purpose global analysis program developed at the Laboratory for Fluorescence Dynamics (LFD). Overall flow diagrams as well as specific FORTRAN 77 source codes of key programming sections are ... [truncated at 150 words]

Introduction: Spontaneous, microscopic fluctuations are an integral part of every fluorescence measurement and add a noise component to the observed fluorescence signal.Fluorescence correlation spectroscopy (FCS) extracts information from this noise and characterizes the kinetic processes that are responsible for the signal fluctuations. For instance, the dynamic equilibrium between a fluorescent and a nonfluorescent state of a fluorophore introduces fluctuations. Another example is Brownian motion, which leads to the stochastic appearance and disappearance of fluorescent molecules in a small observation volume. FCS characterizes any kinetic process that leads to changes in the fluorescence, because the spontaneous fluctuations at thermodynamic equilibrium are governed by the same laws that describe the kinetic relaxation of a system to equilibrium. Thus, FCS offers a very convenient method for determining kinetic properties at equilibrium without requiring a physical perturbation of the sample. This is especially important for systems in which the use of perturbation techniques in ... [truncated at 150 words]

We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation. Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation–and therefore affect the measured fluorescence lifetime–that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging. FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging ... [truncated at 150 words]

Introduction: Phospholipase A2 (PLA2) catalyzes hydrolysis of the sn-2 acyl chain of phospholipids. Physiologically, it appears to be involved in diverse functions such as digestion, membrane homeostasis, production of precursors for synthesis of several lipid mediators, defense against bacteria, clearing of dead or damaged cells, and as ligands for receptors. Three basic typeshave been identified: secretory, cytosolic, and intracellular PLA2 (sPLA2, cPLA2, and iPLA2, respectively)...

2002

Tramier M, Holub O, Croney JC, Ishii T, Seifried SE, Jameson DM.

Binding of ethidium to yeast tRNA(Phe): a new perspective on an old Bromide.

We have reinvestigated the binding of ethidium bromide (EB) to yeast tRNA(Phe) using frequency domain fluorometry and Global Analysis. Previous fluorescence investigations of EB-tRNA interactions, carried out for more than 30 years, have indicated a "strong" binding site with a lifetime near 26 ns and one or more "weak, non-specific" binding sites with reduced lifetimes. In our study, under specific conditions in which only one EB is bound, a fluorescence lifetime of 27 ns was obtained. However, as the EB/tRNA ratio increased, shorter lifetime components appeared. Global Analysis of the lifetime data was consistent with a model in which the second EB molecule bound has a lifetime of only 5.4 ns. Global Analysis also indicated that this second binding event leads to a reduction in the lifetime of the first EB bound, namely from 27 ns to 17.7 ns. The lifetime decrease associated with the "strong" binding site could be ... [truncated at 150 words]

Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of sub-populations. Yeast membranes are a primary site of environmental response and adaptation. Using fluorescence spectroscopy with DPH polarization and laurdan Generalized Polarization (GP), we previously found rapid “average” membrane fluidity modulation in yeast populations during growth and in response to nutrients or environmental stresses. To determine whether such responses reflect all cells we conducted the first multi-photon scanning fluorescence microscopy study of yeasts, measuring laurdan GP. We assessed membrane fluidity responses of individual yeasts related to growth phase, heat stress and ethanol stress. Average fluidity decreased as cultures aged, however the decreased fluidity was due in some cases to an increasing proportion of uniformly ... [truncated at 150 words]

2001

Dong CY, Buehler C, So PTC, French T, Gratton E.

Biological applications of time-resolved, pump-probe fluorescence microscopy and spectroscopy in the frequency domain.

In biological applications of optical microscopy, technical developments often lead to novel imaging modalities with significant applications. For example, the development of confocal microscopy led to microscopic imaging with enhanced contrast (see Chapter 5), and the more recent development of two-photon fluorescence microscopy revolutionized fluorescence microscopy by providing an imaging modality capable of high image contrast, reduced photodamage, and exciting possibilities in controlling localized photochemical reactions in three dimensions (see Chapters 9-13).

Two-photon excitation spectroscopy has inherent 3-D resolution with excitation volumes as small as 0.1 fl. Compared to conventional fluorometers a reduction by a factor of 10^10 of the excitation volume can be achieved. The fluorescence fluctuations within the small excitation volume provide, via fluorescence correlation spectroscopy (FCS), a unique way to study biological phenomena. In this contribution, we outline the instrumentation of two-photon fluorescence correlation spectroscopy and highlight technical details of our experimental setup. We discuss the autocorrelation analysis for single and multiple species with emphasis on the normalized fluctuation amplitude G(0). Furthermore, we revisit another data analysis technique called moment analysis. This method was originally introduced 10 years ago and provides a very fast and convenient characterization of the fluctuation amplitude. We compare experimental results from moment analysis with that of another data analysis method, the photon counting histogram (PCH), and discussed differences between these two methods.

In the macroscopic world fluctuations are typically exceedingly small and beyond the resolution power of most experiments. Only in special circumstances, such as near the critical point of a liquid, do fluctuations become visible to the naked eye. However, the importance of fluctuations increases once the macroscopic world is left behind and one starts to consider mesoscopic or microscopic systems, where fluctuation phenomena are readily observed. Fluctuation spectroscopy exploits this source of information [20.1] and embraces a diverse field of applications.

Breusegem SY, Loontiens FG, Regenfuss P, Clegg RM.

Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques.

One naturally needs to expand from classical single-point measurement to simultaneous multiple-point measurement for the formation of an image. This technical expansion raises a new challenge, namely, the ability to acquire images with fluorescence lifetime information within a reasonable time frame and with similar accuracy and precision comparable to that of single-point spectroscopy measurement. This goal is the main scope of this chapter, which provides an overview of the different techniques used in fluorescence lifetime imaging microscopy (FLIM).

This chapter begins with an overview of two-photon microscopy relevant to applications in deep-tissue imaging. Basic principles, deep-tissue models, and instrumentation are discussed. From there, recent advances in two-photon deep-tissue imaging are addressed: (1) the application of a blind deconvolution algorithm in further improving image quality and providing point spread function (psf) information in deep tissue, (2) the development of video-rate two-photon instrumentation to extend the imaging technology to potential clinical applications, and (3) the acquisition and analysis of two-photon spectroscopic data as complementary information to structural imaging.

1999

Masters BR, So PTC, Gratton E.

Multiphoton excitation microscopy and spectroscopy of cells, tissues, and human skin in vivo.

This chapter describes the history, theory, instrumentation, and cell and tissue applications of non-linear multiphoton microscopy. It includes a discussion of laser sources, detectors, scanners, and microscope objectives, and experimental applications are presented to illustrate the unique capabilities of these innovative techniques. The emphasis of the chapter is on the application of multiphoton excitation microscopy to the functional imaging of in vivo human skin, and the two examples presented—of transparent and turbid media—illustrate the utility of multiphoton excitation microscopy in thick specimens. Multiphoton excitation microscopy is an important new optical technique with many applications in biology. It has advantages over confocal laser scanning microscopy as well as wide-field microscopy. The high peak power of the laser pulses can result in cell damage in highly pigmented cells, and this damage can occur even with a single pulse of light. Multiphoton excitation microscopy can be exploited to capitalize on its improved penetration, ... [truncated at 150 words]

Masters BR, So PTC, Kim KH, Buehler C, Gratton E.

Multiphoton excitation microscopy, confocal microscopy, and spectroscopy of living cells and tissues; functional metabolic imaging of human skin in vivo.

Fluorescence is an especially sensitive technique that is a vital contrast enhancement mechanism for microscopy. Fluorescence microscopy has excellent background rejection and highly specific staining (particularly with the use of antibody-conjugated probes). It provides the high contrast needed to spatially resolve microscopic structures such as cellular organelles. Although the spatial relationship of the organelles is important, the functional properties of the organelles are also vital to the understanding of cellular life. Local properties such as pH or molecular concentration can reveal much about the organelle. Fluorescent probes are normally very sensitive to the local environment. For example, some probes are fluorescent only in a particular pH or polarity condition. Fluorescence intensity measurements, however, are unsuitable for quantitative work as the measured intensity is not just dependent on the environment but also dependent on the local probe concentration which cannot easily be determined. Quantitative measurements require a parameter that is independent ... [truncated at 150 words]

Two-photon excitation microscopy has the potential as an effective, noninvasive, diagnostic tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue scattering and absorption coefficients in the infrared wavelengths. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multiphoton excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 200 microns. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical ... [truncated at 150 words]

Time-resolved pump-probe spectroscopy is routinely used to study ultrafast phenomena in biological systems. Important systems such as photosynthetic reaction centers, rhodopsins, and hence proteins exhibit Fictionally important processes on femtosecond and picosecond time scales. This chapter describes the application of pump-probe spectroscopy techniques to time-resolved microscopy. In addition to the well-known advantages associated with time-resolved microscopy pump-probe microscopy can facilitate functional studies of ultrafast protein reactions and motions in native environments (i.e., cells and tissues).

Gratton G, Fabiani M, Corballis PM, Gratton E.

Noninvasive detection of fast signals from the cortex using frequency-domain optical methods.

INTRODUCTION: The study of the functioning of the human brain has recently become one of the most significant areas in science, as demonstrated by the official declaration of the 1990s as the "Decade of the Brain." A considerable amount of research has focused on the study of physiological phenomena associated with sensory, cognitive, and motor functions. Theories in these areas emphasize that these functions depend on the dynamic interaction of different brain areas.The last few years have also seen a considerable expansion in the physiological methods used to examine the functioning of the human brain. By and large, two major classes of techniques have evolved: techniques measuring the electromagnetic fields produced by active neurons (such as event-related potentials-ERPs-and magnetoencephalography-MEG), and techniques measuring hemodynamic or metabolic changes that are associated with neuronal activity (such as positron emission tomography-PET-and functional magnetic resonance imaging-fMRI). The types of information that are derived from these ... [truncated at 150 words]

A new, powerful fluorescence microscope has been developed by combining two-photon excitation and fluorescence time-resolved imaging. Scanning microscopy with two-photon excitation is an alternative to conventional confocal scanning for studying the internal structure of cells (1-3). This method produces a three-dimensional (3-D) sectioning effect similar to that of conventional confocal microscopy, but with additional advantages: (1) photo-bleaching and photodamage are localized; (2) samples labeled with UV probes can be visualized without UV lasers or expensive quartz optics; and (3) superior background rejection can be obtained. Fluorescence time-resolved measurements provide an additional contrast-enhancing mechanism for microscopic imaging (4-10). Multiple labeling of cellular organelles using fluorescent probes with similar excitation/emission spectra but different lifetimes can be distinguished by time-resolved methods. Lifetime measurements can also monitor the local environment of fluorescent probes, such as cellular calcium concentration and oxygen distribution.In this chapter. we present the fundamentals of two-photon scanning , microscopy for both ... [truncated at 150 words]

In this chapter we will describe the characteristic properties of a number of lasers commonly used in fluorescence microscopy. We will concentrate on the characteristics of lasers in relation to their use as an illumination source. Lasers have a number of unique properties compared to other sources emitting electro-magnetic radiation, such as arc lamps, which make them an almost ideal light source for use in confocal microscopy. These properties are:* high degree of monochromaticity* small divergence* high brightness* high degree of spatial and temporal coherence* plane polarized emission (for many types)* Gaussian beam profile (can be obtained by special optics)Over the last 30 years, since the realization of the first experimental laser, a wide and still expanding variety of lasers has been developed. Available laser systems cover an extremely wide spectrum, differing from each other in physical size, principle of operation, optical, temporal and mechanical properties, such as beam divergence, ... [truncated at 150 words]

INTRODUCTION: The kinetic characterization of catalysis by enzymes entrapped in reverse micelles of amphiphiles in organic solvents has received a great deal of attention. Reverse micelles can be formed by phospholipids or detergents and are usually capable of taking up relatively large amounts of water in the internal cavity, forming water pools dispersed in the organic phase. Several hydrophilic, water soluble enzymes retain enzymatic activity in reverse micelles, which presents interesting questions regarding their mechanisms of catalysis and the possible role of solvent in their function.

Fluorescence methods are widely used in a number of research fields, in both industry and medicine. The instrumentation employed for the measurement of fluorescence parameters varies according to whether steady-state or time dependent measurements are performed. The cost of the fluorescence instrumentation and the complexity of its use are major factors in determining the kind of measurement to be performed in relation to a particular problem. In principle, time-resolved measurements contain more information than steady-state measurements, since the steady-state values represent the time average of the time-resolved determinations. However, the instrumentation used to perform lime-resolved measurements is usually more complicated, and, traditionally, it is used only by experts in the field. Also the sensitivity afforded by time-resolved fluorescence measurements is usually less than the sensitivity of steady-state measurements. To obtain an adequate time resolution. mode-locked picosecond pulsed lasers are employed. Alternatively, frequency-domain methods can be used. One of the factors ... [truncated at 150 words]

Proteins are dynamic and not static systems; their internal motion is crucial for their function. Water is essential for protein motions and consequently also for protein function. Beyond these general statements, a full understanding of the relation between motion and structure and between hydration and motions is not yet available, and most of the essential features of the role of water for biological action remain to be elucidated. In this chapter we outline the general aspects of protein states and protein motions and discuss how water may be involved. This chapter is not a comprehensive guide to dynamics and hydration, but a modest road map to further work. To be specific, we will explain concepts and processes by using simple examples, but we believe that the phenomena are general and occur in various disguises in most proteins and probably also in nucleic acids.Protein dynamics has been treated in a number ... [truncated at 150 words]